The present invention relates to a method and assay for determining a level of an acyl coenzyme A ester or esters in a sample. The method and/or assay finds particular application in screening and prediction of human fatty acid oxidation disorders.
Measurement of acyl coenzyme A (CoA) esters may have important utility in the diagnosis of human fatty acid oxidation (FAO) disorders. Up to 5%; of sudden infant death occurrences are likely to be a consequence these disorders (Boles et al. 1998).
The most common FAO disorder in medium chain acyl CoA dehydrogenase (MCAD) deficiency, which may affect up to 1 in 6400 new-borns in the UK (Eaton et al. 1996). MCAD deficiency is easily treated by avoiding fasting; however there is no currently accepted screening protocol for this disease. Universal screening for MCAD in the UK has been recommended (Bonnet et al. 1998, Clayton et al. 1998, Pollit and Leonard 1998). Other conditions where fatty acid metabolites may be problematic include the remission of protein energy malnutrition (PEM) in infants, where toxic acyl CoA compounds may accumulate and contribute to worsening of patient condition (Terangarcia et al. 1998).
FAO disorders result in a variety of abnormal urinary and blood plasma metabolite levels; however screening is conventionally carried out on tissue biopsies (Eaton et al. 1996). This makes wide-scale neonatal screening by this method impractical. MCAD deficiency and other FAO disorders are currently diagnosed by the generation and measurement of radiolabeled acyl CoA products with atypical chain lengths from radiolabeled precursor. An alternative diagnostic procedure is to use the polymerase chain reaction (PCR) technique to identify the relevant gene(s), but this is only 85 to 90% accurate (Eaton et al. 1996). Diagnosis of MCAD deficiency and other FAO disorders would benefit most from the application of simple methods to routinely and directly measure changes in the concentrations of plasma and urine metabolites, such as acyl CoA esters.
Routine monitoring of acyl CoA ester concentrations in tissues has not been attempted because of the absence of suitable low cost, high throughput screening methodologies. Traditional detection methods for these compounds utilize high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) absorption. This method can detect down to approximately 500-1000 fmoles of acyl CoA esters. This method is not however sensitive enough to reliably detect changes in ratios of acyl CoA esters associated with FAO disorders. A newer technique, which involves glycine aminolysis, pentafluorobenzyl bromide esterification, and gas chromatography/mass spectrometry can detect acyl CoA esters at concentrations as low as 30 fmoles (Tamvakopoulous and Anderson 1992). However, this method is time-consuming, complex, and requires expensive kit.
It is amongst the objects of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages.
In a first aspect the present invention provides a method for measuring an acyl coenzyme A (acyl CoA) ester or esters in a sample, comprising the steps of:
a) forming a reaction mixture comprising the sample to be tested and a derivatizing agent;
b) allowing the sample and said derivatizing agent to react, so as to form a fluorescent derivative(s) of any acyl CoA ester(s) present in the sample; and
c) determining a level of said fluorescent derivative(s).
It is to be understood that the acyl CoA esters of the present invention are of the form where a fatty acid is linked to coenzyme A by way of a thioester linkage between the carboxyl group of the fatty acid and the sulfhydryl group of CoA. Fatty acids are in fact typically oxidised by first forming such an acyl CoA ester as described and thereafter sequential rounds of degradation by acyl CoA dehydrogenases which have different acyl chain-length specificity.
The method may be used for example to measure levels of acyl CoA esters present in plant tissue where it could have application in improving yield of unusual fatty acids in oil seed crops engineered to produce the same. However, the method finds particular application in measuring levels of acyl CoA esters in samples obtained from an animal and subsequent determination of any possible fatty acid oxidation (FAO) disorders in the animal.
Thus, in a second aspect the present invention provides an assay for determining a level of an acyl coenzyme A (acyl CoA) ester or esters in a test sample, comprising the steps of:
a) obtaining the test sample from an organism to be tested;
b) forming a reaction mixture comprising the sample to be tested and a derivatizing agent;
c) allowing the sample and said derivatizing agent to react, so as to form a fluorescent derivative(s) of any acyl CoA ester(s) present in the sample;
d) determining a level of said fluorescent derivative(s) ester(s); and
e) comparing the level of said fluorescent derivative(s) in the test sample with a level of said fluorescent derivative(s) in a normal sample, such that a significant difference in the level of said fluorescent derivative(s) between the test sample and normal sample may be predictive of a fatty acid oxidation disorder in said organism.
The organism to be tested may be any suitable organism which metabolises acyl CoA esters. However the assay is particularly suited to testing animals, such as cows, sheep, dogs, cats, goats, pigs, horses and especially humans. It is envisaged that the assay may be used to test newborns in order to determine if the newborn has any fatty acid oxidation disorder, such as MCAD deficiency, or any other disorder which results in alteration of acyl CoA levels, such as protein energy malnutrition. Fatty acid oxidation disorders may be associated with an accumulation or depletion of certain acyl CoA esters in comparison to normal levels.
The test sample may be any sample in which acyl CoA esters are present. For example the test sample may be a sample of tissue taken from the organism. However, it is preferred that the sample to be tested is obtained from the organism with the minimum of distress or discomfort to the organism. Thus, for animals the preferred source of the sample is from the blood, eg. plasma, or urine. A urine sample is particularly preferred since obtaining the sample is non-invasive. other samples may include samples from tears, nasal or vaginal swabs.
The derivatizing agent may be any agent which upon reaction with an acyl CoA renders the acyl CoA sufficiently fluorescent such that the fluorescent acyl CoA derivative may be detected at low concentrations. Typically the fluorescent acyl CoA derivative may be detected at concentrations less than 100 pmoles, for example less than 50 pmoles, such as less than 5 pmoles. The presently described method may even be used to detect acyl CoA esters in the 10s of fmol range. The skilled reader will appreciate that the sensitivity of the present method will depend to some extent on the derivatizing agent employed and the degree to which the acyl CoA is rendered fluorescent.
It may be possible to form fluorescent derivatives of acyl CoA esters with a number of amino reactive probes, amino derivatization reagents or protein labelling reagents. These reagents may act to modify the amine group on the coenzyme A adenine ring to form fluorescent derivatives. These may include, but are not limited to the commonly used reagents chloroacetaldehyde, dansyl or dabsyl chloride, napthalens-2,3-dicarboxyaldehyde (NDA), fluorescein, and o-phthaldialdehyde (OPA).
Generally speaking the derivatizing agent may react with the amine group on the coenzyme A adenine ring to form the fluorescent derivative(s). For example, the present inventors have found that the use of chloracetaldehyde as the derivatizing agent results in the amine being derivatized with the formation of an etheno group.
The fluorescent derivatives of the acyl CoA esters may be conveniently detected using a fluorometer. Typically there will be a number of acyl derivatives in any particular sample and in order to accurately determine their presence it is also necessary to separate them in some manner. Thus, separation may be preformed using HPLC techniques commonly known in the art with the fluorometer coupled to the HPLC to detect the fluorescence of the acyl CoA derivatives.
It is immediately evident to one skilled in the art that in order to determine if the level of an acyl CoA in a test sample is abnormal or deficient it is necessary to compare the level determined in the test sample with a control and/or normal sample. Thus, in accordance with the assay a control sample, comprising known amounts of particular acyl CoA esters having varying length acyl groups (eg. C2-C16) may be run in conjunction with the test sample in order to allow identification and quantification of the acyl CoA esters in the test sample. Additionally, or optionally, a normal sample may also be run with the test sample. A normal sample would be a sample from a healthy organism which does not have a fatty acid oxidation disorder. This would allow comparison of the normal and test sample results and determination of whether or not any level of acyl CoA in the test sample is significantly different to a normal sample.
It is expected that, for example, a subject displaying an MCAD deficiency may have nigher levels of medium chain acyl CoA esters (eq. C8-C12) in comparison to a normal sample.
In a further aspect there is provided a kit for use with the assay of the present invention, the kit comprising
a derivatizing agent for fluorescently derivatizing an acyl CoA ester(s) present in a test sample; and
a normal and/or control sample for comparing the level of said acyl CoA esters(s) in the test sample with the level of acyl CoA ester(s) in the normal and/or control sample.
The normal and/or control sample(s) may already be derivatized or may be derivatized in conjunction with the test sample by the derivatizing agent.